Bioelectric method for the detection of anticholinester-ases



Sept. 27, 1966 D. L. CANNON, JR., ET AL 3,275,534

BIOELECTRIC METHOD FOR THE DETECTION OF ANTICHOLINESTERASES Filed March19, 1965 HIGH IMPEDENCE VTVM RECORDER 4.0 2 I15 I- 3.0 3 2 g 2.0-

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United States Patent BIOELECTRIC METHUD FOR THE DETECTKON 0FANTlCHOLINESTER-ASES Paul L. Cannon, in, Harrisburg, Pa., and David N.Kramer, Stevenson, Md, assignors to the United States of America asrepresented by the Secretary of the Army Filed Mar. 19, 1963, Ser. No.266,461 Claims. (Cl. 204-11) The invention described herein may bemanufactured and used by or for the Government of the United States ofAmerica for governmental purposes without the payment to us of anyroyalty thereon.

This invention relates to a new method for the detection ofanticholinesterases. More specifically the present invention providesfor the electrical monitoring of cholinesterase catalyzed reactions.

One of the advantages of the present invention is that it provides anaccurate laboratory method for the remote deterimination of microamounts of a highly toxic agent in a manner similar to the method setforth in the patent issued to Northrop 2,757,132.

Some of the organophosphorous compounds are highly toxicanticholinesterase agents. One of the more important enzymes in thehigher animals is cholinesterase which is vital for the transmission ofnerve impulses for muscle control. Minute amounts of anticholinesteraseagents such as the G agents, which are disclosed in Patent 2,926,072,inactivate this enzyme and cause paralysis of the animals muscularsystem and eventual death from failure of the respiratory system.

It is an object of this invention to detect amounts of theseorgano-phosphorous anticholinesterase agents in! the order ofrnillionths of a gram per milliliter of solution. It is a further objectof this invention to detect the above minute amounts of theseanticholinesterase agents with an accuracy of about 11%.

It has been proposed (US. Patent 3,049,411) to detect these agents bymeans of the reaction of cholinesterase with colorless acetates ofindophenols. In the absence of an agent, the indophenolester ishydrolyzed to a highly colored compound. The present method is adistinct improvement over this patented method since it gives aquantitative indication of the presence of such agents and is notinterfered with by extraneous colored solutions.

The above objects are achieved in this invention by applying a directcurrent across two platinum electrodes immersed in a bufferedapproximately neutral aqueous solution of the anticholinesterasecompound and a thiocholine ester which also has a half cell or areference electrode therein. The potential between the positiveelectrode and the reference electrode is then recorded as a function oftime. Cholinesterase is then added to the solution and the time ofaddition is noted. There will be a change in the recorded potential as afunction of time. This potential change per unit of time is the rate ofdepolarization of the positive electrode caused by This change inpotential per unit of time or voltage-time is linearly related to theconcentration of the enzyme. In the presence of anticholinesterase theactivity of the enzyme is decreased as a function of inhibitorconcentration. It has been found that the ratio is inverselyproportional to the concentration of the inhibitor. Thus, a relativelylarge ratio indicates a relatively small amount of inhibitor present inthe solution.

The thiocholine esters which can be used in this process are the acetyl,propionyl, and butyryl thiocholine iodides or the lower alkanoyl estersof ethanthiol trimethyl ammonium iodide. The pH of the solution in theconcentration cell must be approximately neutral, i.e., in the range ofpH from 68 with the preferred pH being 7.4.

In the drawing, FIG. 1 illustrates the electrical circuit with thevacuum tube volt meter and the recorder connected to the solution.

FIG. 2 shows the voltage-time curves for the enzymatic hydrolysis ofbutyrylthiocholine iodide by cholinesterase inhibited by; in case A, noagent; in case B, 0.36 g. per ml.; in case C, 0.50 g. per ml.; in caseD, 1.0 g. per ml. of isopropyl methyl phosphonofiuoridate (hereinafterdesignated as GB), respectively.

FIG. 3 shows the calibration plots of AE/At vs. the agent concentrationin ig/ml. for four typical anti-cholinesterase agents.

In FIG. 1, the apparatus used to apply a constant low amperage currentand to measure the rates of depolarization is shown. A -volt battery isapplied across two platinum thirnble electrodes, 17 and 19 through aresist ance of a 270-kilohm resistor, 11, and a Z-megohm resistor, 15.The circuit is stabilized by means of a Zener diode, 13. A referencehalf cell such as a saturated calomel electrode is shown at 21. Thesolution is contained in a concentration cell, 23, which is constantlystirred and maintained at a constant temperature by means of a standardwater jacket or by location in a constant temperature chamber.

Example 1 Twenty-five ml. of a 1 10- M butyrylthiocholine iodide in 0.1M aqueous tris(hydroxymethyl)aminomethane solution (pH 7.40) is placedin a 50-ml. beaker, and 1.0 ml. of an aqueous GB sample is added. Thissample can contain 1 to 15 ,ug. of GB or the agent which is to beanalyzed. The resulting solution is magnetically stirred at atemperature of 25 C., the platinum electrodes and the referenceelectrode are immersed into the solution and a current of 25 ,MOL isapplied across the two platinum electrd-oes by means of the circuitshown in FIG. 1. The recorder is switched on, and the resultingpotential of the platinum anode vs. the reference electrode isautomatically recorded. At zero time, 1.0 ml. of a 1.0 mg. per ml.cholinesterase solution is added to effect the enzymatic hydrolysis ofthe substrate.

Under operating conditions without the addition of GB, addition ofcholinesterase to the magnetically stirred solution of the thiocholineester caused a rapid hydrolysis of the substrate to thiocholine andbutyric acid. The thiocholine produced causes a depolarization of theelectrodes and a reduction in the potential of the system. This isillustrated by the aforementioned curves of FIG. 2. By means ofpredetermined calibration plots of the type set forth in FIG. 3 one canobtain the amount of agent present in the original sample. In a mannersimilar to Blaedel and Hicks (Analytical Chemistry, vol. 34, No. 3,March 1962, pages 388-394), one can calibrate the recorder to give adirect readout in micrograms of the agent. The temperature must beclosely controlled to give the optimum results as far as accuracy isconcerned.

We have found that the above procedure works well with GB and Systox(0,0-diethyl-O-[Z-(ethylthio)-ethyl] phosphorothionate). However, forcompounds such as parathion (diethyl-p-nitrophenyl thiophosphate) andmalathion (S-[1,2-dicarbethoxyethyl]-0,0 dimethyl-dithiophosphate), wehave found that these compounds must be preincubated wtih the enzyme forabout ten minutes to give sensitive and accurate results. Likewise, withGB or Systox a short incubating period of 3-5 minutes made the processmore sensitive to smaller amounts of these agents.

We have also found that with parathion and malathion and other waterinsoluble agents a cosolvent had to be used to get these agents into thesolution We have found that an aliphatic oxygenerated solvent such asglycerol, ethylene glycol, diethylene glycol, diethylene glycolmonoethyl ether, ethylene glycol monomethyl ether were excellentcosolvents to use in our process Best results were obtained with the useof ethylene glycol monomethyl ether. It was found that up to about 1% ofthe aliphatic oxygenated solvent could be tolerated in the finalreaction mixture without giving erratic results.

Our method can be applied to other agents or inhibitors such as GA, GD,paraoxon, malaoxon, and DFP with equally good results.

We claim:

1. A method of quantitatively detecting the presence of organophosphoruscompounds which comprises:

(a) applying a low direct current across a negative and a positiveelectrode immersed in a stirred buffered aqueous solution of saidcompound and a lower alkanoyl thiocholine ester having a referenceelectrode therein,

(b) recording the potential between said positive electrode and saidreference electrode,

(c) adding cholinesterase to said solution,

(d) recording the ratio of the change in potential between said positiveelectrode and said reference electrode with respect to the change intime from the addition of said cholinesterase, said ratio beinginversely proportional to the concentration of said compound in saidbuffered solution.

2. A method of quantitatively detecting the presence of organophosphoruscompounds which comprises:

(a) applying a weak direct current across a negative and a positiveelectrode immersed in a neutral aqueous solution of said compound and alower alkanoyl thiocholine ester which has a reference electrodetherein,

(b) recording the potential between said positive electrode and saidreference electrode,

() adding cholinesterase to said solution,

((1) recording the ratio of the change in potential between saidpositive electrode and said reference electrode with respect to thechange in time from the addition of said cholinesterase, said ratiobeing inversely proportional to the concentration of said compound insaid neutral solution.

3. A method of quantitatively detecting the presence of isopropyl methylphosphonofluoridate which comprises:

(a) applying a direct current of low amperage across a negative and apositive electrode immersed in a stirred buffered approximately neutralaqueous solution of said phosphonofluoridate and a lower alkanoylthiocholine ester having a reference electrode therein,

(b) recording the potential between the positive electrode and saidreference electrode,

(c) adding cholinesterase to said solution,

' ((1) recording the ratio of the change in potential between saidpositive electrode and said reference electrode with respect to thechange in time from the addition of said cholinesterase, said ratiobeing inversely proportional to the concentration of saidphosphonofluoridate in said buffered solution.

4. A method of quantitatively detecting the presence of water solubleorganophosphorus compounds which comprises:

(a) applying a weak direct current across a negative and a positiveelectrode immersed in a stirred aqueous addition of said cholinesterase,said ratio being inversely proportional to the concentration of saidorganophosphorus compound in said solution. 5. A method as set forth inclaim 4 in which the organophosphorus compound is isopropyl methylphosphonofluoridate.

6. A method of quantitatively detecting the presence of water insolubleorganophosphorus compounds which comprises:

(a) applying a weak direct current across a negative and a positiveelectrode immersed in a stirred aqueous solution containing saidcompound, tris(hydroxymethyl)aminomethane, butyrylthiocholine iodide,and up to about 1% of the total solution of a solvent selected from thegroup consisting of glycerol, ethylene glycol, diethylene glycol,diethylene glycol monoethyl ether, and ethylene glycol monomethyl ether,said solution being adjusted to a pH range of 7.0 to 8.0 and having areference electrode therein,

(b) recording the potential between said positive electrode and saidreference electrode,

(c) adding cholinesterase to said solution,

((1) recording the ratio of the change in potential between saidpositive electrode and said reference electrode with respect to thechange in time from the addition of said cholinesterase, said ratiobeing inversely proportional to the concentration of saidorganophosphorus compound in said solution.

7. A method as set forth in claim 6 in which the solvent is ethyleneglycol monomethyl ether.

8. A method as set forth in claim 7 in which the organophosphoruscompound is 0,0-diethyl O-2-(ethylthio)-ethyl phosphorothionate.

9. A method as set forth in claim 7 in which the organophosphoruscompound is diethyl-p-nitrophenyl thiophosphate.

10. A method as set forth in claim 7 in which the organophosphoruscompound is S-(1,2-dicarbethoxyethyl)- 5O 0,0-dimethyldithiophosphate.

References Cited by the Examiner UNITED STATES PATENTS 2,757,132 7/1956Northrop 204195 2,926,072 2/1960 Kramer et al. 23232 3,049,411 8/1962Gelman et al 23232 OTHER REFERENCES Blaedel et al.: AnalyticalChemistry, vol. 34, No. 3, March 1962, pp. 388394.

JOHN H. MACK, Primary Examiner.

T. H. TUNG, Assistant Examiner.

1. A METHOD OF QUANTITATIVELY DETECTING THE PRESENCE OF ORGANOPHOSPHROUSCOMPOUND WHICH COMPRISES: (A) APPLYING A LOW DIRECT CURRENT ACROSS ANEGATIVE AND A POSITIVE ELECTRODE IMMERSED IN A STIRRED BUFFERED AQUEOUSSOLUTION OF SAID COMPOUND AND A LOWER ALKANOYL THIOCHOLINE ESTER HAVINGA REFERENCE ELECTRODE THEREIN, (B) RECORDING THE POTENTIAL BETWEEN SAIDPOSITIVE ELECTRODE AND SAID REFERENCE ELECTRODE, (C) ADDINGCHLORINESTERASE TO SAID SOLUTION, (D) RECORDING THE RATIO OF THE CHANGEIN POTENTIAL BETWEEN SAID POSITIVE ELECTRODE AND SAID REFERENCEELECTRODE WITH RESPECT TO THE CHANGE IN TIME FROM THE ADDITION OF SAIDCHLORINESTERASE, SAID RATIO INVERSELY PROPORTIONAL TO THE CONCENTRATIONOF SAID COMPOUND IN SAID BUFFERED SOLUTION.